The Ipsos Mori poll of 1,003 Scots showed support for the union at 58% and support for independence at 30% among those certain to vote in the referendum.
Asking the Scottish Government's preferred question: 'Do you agree that Scotland should be an independent country?', the results follow two other polls by the same company earlier this year, showing a trend in opinion.
Opposition to independence increased from 50% in January to 55% in June then 58% in the latest poll, while support for independence dropped from 39% to 35% then 30%.
Last week a TNS-BMRB poll in The Herald showed opposition to independence surging, with supporters of the Union opening up a 25% lead over the pro-independence Yes campaign.
Christopher McLean, senior research executive with Ipsos Mori Scotland, said today: "Now that the process for the referendum has been agreed and the real debate can begin, this poll highlights the considerable challenges facing the Yes campaign.
"At the turn of the year, support for independence was increasing and touching 40%. That momentum appears to have been lost and the level of support for independence has returned to its historical average.
"On the other hand, although the 'Better Together' campaign has a healthy lead, the poll shows that they cannot afford to be complacent, particularly given that more than one in ten Scots remain undecided."
The poll, published in The Times today, shows Alex Salmond remains the most popular leader in Scotland with half of Scots satisfied with his performance, but there has been an increase in those dissatisfied with the First Minister over the past year.
His net satisfaction rating - the proportion of those satisfied minus the proportion of those dissatisfied - has fallen from plus 35% in December last year to plus 10% in Ipsos Mori's latest poll.
Scottish Labour leader Johann Lamont has also seen her net satisfaction rating fall since June, with 35% satisfied with her performance as leader while 30% are dissatisfied.
In voting intentions for the Scottish Parliament, the SNP has 40% support, a 5% fall since June, and Labour has 35% support, an increase of three points over the same period. The figures show a ten-point swing in favour of Labour since December 2011, according to Ipsos Mori.
The Conservatives remain in third place on 13% with the Liberal Democrats in fourth on 8%.
Deputy First Minister Nicola Sturgeon said: "We are very confident of achieving a Yes vote in autumn 2014, because we know from other surveys that a clear majority of people in Scotland believe that the Scottish Government is better at making decisions for Scotland than Westminster - by 64% to 24% - which is an essential foundation of the case for an independent Scotland.
"By spelling out over the next two years how an independent Scotland will flourish - and that the alternative is to have the achievements of home rule such as free personal care and no tuition fees rolled back - I believe that we will turn this potential majority into a Yes majority in 2014.
"It is also fantastic to see the SNP retain a strong opinion poll lead after over five years in government. And with these positive ratings of 'plus 10', the First Minister remains far and away the most popular political leader in the UK."